Neospora caninum and Toxoplasma gondii are closely related protozoan parasites responsible for disease in a wide range of animals. N. caninum has recently been recognised as an important cause of neuromuscular disease in dogs, and as a cause of abortion and neonatal mortality in cattle and other domestic animals. T. gondii is a common cause of ovine abortion, and is one of the most important opportunistic pathogens in immunosuppressed human patients, such as those with acquired immune deficiency syndrome. Effective management of neosporosis and toxoplasmosis requires prompt diagnosis and treatment. The development of a range of efficient diagnostic tests is therefore essential.
Diagnosis of neosporosis and toxoplasmosis is difficult due to the vague nature of early clinical signs and the low numbers of parasites present in infected tissues. In the past, neosporosis was misdiagnosed as toxoplasmosis because of the similarities in clinical signs and pathological changes associated with infection with each organism. While serological and immunohistochemical techniques may aid diagnosis, clinically normal animals may have antibody titres suggestive of disease, and interpretation of immunohistochemistry is sometimes difficult due to variable staining and cross-reactivity. Molecular biological techniques such as the polymerase chain reaction (PCR) offer a highly sensitive and specific alternative to immunologic approaches to diagnosis. Previously developed PCR protocols for N. caninum and T. gondii, however, have not fully exploited the potential of this technique.
The PCR protocols developed by the present inventors utilize primers which hybridise within the internal transcribed saucer 1 (ITS1) region of the ribosomal RNA (rRNA) gene Unit of N. caninum and T. gondii (Payne and Ellis, 1990). The ITS1 region of the rRNA gene unit is relatively variable but also contains conserved regions consistent with its role in processing the rRNA molecule. Both the ribosomal RNA and the transcribed spacer regions have significant secondary structure due to their rule in protein translation and in the processing of the rRNA molecules respectively.
In the absence of a species-specific gene, the ITS1 region maybe a target for diagnostic PCR, as it is present at high copy number and exhibits high inter-species variability, while being generally conserved within a species Payne and Ellis, 1996). Furthermore, the ITS1 is readily sequenced and characterized because it is flanked by the 18S and 5.8S rRNA genes. The ITS1 regions of N. caninum and T. gondii are 421 and 392 bp, respectively, and the similarity between the two species is 82% (Payne and Ellis, 1996). Guay and co-workers (1993) estimated there to be around 110 copies of the ribosomal RNA gene unit in each T. gondii genome.
High sensitivities have been reported for PCR protocols targeting the ribosomal RNA gene unit, due to the high gene copy number. Guay and co-workers (1993) described a PCR capable of detecting a single organism using primers specific for the ITS1 region of T. gondii. Holmdahl and Mattson (1995) and Payne and Ellis (1996) recently remand PCR protocols for the detection of N. caninum with was capable of detecting five and seven organisms respectively. While these tests were specific for N. caninum, the sensitivity of the techniques is inadequate for diagnostic PCR because organisms may be present in tissues only in very low numbers and target DNA in some clinical samples may be degraded.
The present inventors have developed a sensitive PCR test for protozoan parasites that is particularly suitable for use in the detection of these microorganisms in clinical and biological samples.